Project summary/abstract: The long-term career goal of Dr. Arunabh Bhattacharya is to establish himself as a successful and well-funded, independent investigator through sustained basic and translational research in the field of aging, in particular, in the area of muscle biology. His doctoral and postdoctoral training in the area of inflammatory diseases and oxidative stress laid the foundation for this career development award application. His research career to date has been highly productive with more than 38 publications in peer- reviewed journals. As a faculty member of the Department of CSB/Barshop Institute for Longevity and Aging Studies in San Antonio, he has been studying the role of lipid mediators generated by the inflammatory pathways in muscle atrophy, which is a new direction of research at UTHSCSA. Barshop Institute has one of the largest Biology of Aging programs in the in the nation and provides excellent opportunities for junior faculty mentoring in aging research. Arunabh is on the path to a long-term independent research career in the field of muscle biology and the K01 career development award will provide the mechanism for him to make this transition. He is submitting this proposal under the joint mentorship of Dr. Van Remmen and Dr. Musi, active faculty members of the Barshop Institute. Dr. Van Remmen is a leading authority in the field of oxidative stress and its role in aging, and Dr. Musi, a funded Ellison Foundation Beeson Scholar, studies the role of inflammatory pathways in skeletal muscle in insulin resistance and aging. The overall training program that has been outlined in this K01 award includes a mixture of practical laboratory training with didactic course work and intensive mentoring interactions with the mentor/co-mentor and other members of the advisory council. In addition, the training program is designed to provide valuable experience in other aspects of research including managing a research budget, running a laboratory independently, forging collaborations and writing grant applications to national funding agencies including Muscular Dystrophy Association and an independent R01 application to National Institute of Aging. Together these factors will ensure his successful transition to an independent investigator in the field of aging and aging-associated changes in quality of life. Through interactions with his mentor/co-mentor, members of the advisory council and the consultants who have agreed to assist Arunabh in this proposal, the K01 award will allow him to gain expertise in a variety of techniques that will provide a strong basis for his continued advancement in the field of muscle biology. Because of his interest in understanding the mechanisms of muscle atrophy during aging, he has designed this K01 grant proposal to study the role of the pro-inflammatory 12/15-lipoxygenase (12/15-LO) pathway in age- related sarcopenia. Based on the preliminary data in mice null for 12/15-LO and in aging mice, he has developed a hypothesis that 12/15-LO plays a significant role in age-related muscle atrophy by modulating the ubiquitin-proteasome and the lysosomal autophagy pathways of protein degradation. This will be tested using a combination of in vitro and in vivo approaches. In Specific Aim 1, in vitro primary myotube culture and isolated muscle preparation will be used to determine the effect of 12/15-LO metabolites, 12- and 15-HETE on the regulation of the ubiquitin-proteasome and lysosomal autophagy pathways. Since induction of protein degradation pathways is modulated by PI3K/Akt signaling and downstream mTOR and FoxO3 pathways, and activation of NF-B, these pathways will be measured in cells and in isolated muscle preparation from wild-type (with/without PI3K/Akt inhibitor) and MISR mice (mice with muscle-specific inhibition of NF-kB) to determine the relative role of PI3K/Akt and NF-B signaling pathways. Activation of downstream lysosomal autophagic pathway will be determined by measuring the expression of autophagic proteins (Atg-7, LC-3II/LC-3I, p62) and the autophagic flux through the pathway and activation of ubiquitin-proteasome pathway by measuring proteasome activity and ability of the proteasome to degrade an ubiquitin-labeled GFP substrate. In Specific Aim 2, the sciatic nerve transection model will be used to determine whether the inhibition of 12/15-LO protects against denervation-induced muscle atrophy by modulating the protein degradation pathways. The level of 12- and 15-HETE, PI3K/Akt signaling and downstream mTOR and FoxO3 pathways, activation of NF-B, markers of the induction of the lysosomal autophagy and ubiquitin-proteasome pathways in control and denervated muscle from wild-type and 12/15-LO null mice will be measured in this Specific Aim. Finally, the hypothesis will be tested in vivo to determine whether inhibition of the 12/15-LO pathway during aging protects against loss of muscle mass and function by modulating the protein degradation pathways. Age-related muscle atrophy and loss of function, level of 12- and 15-HETE, PI3K/Akt signaling and downstream mTOR and FoxO3 pathways, activation of NF-B, and markers of the induction of the lysosomal autophagy and ubiquitin-proteasome pathways in wild-type and 12/15-LO knockout mice and in mice treated with a pharmacological inhibitor of 12/15-LO will be measured in this Specific Aim. In summary, this proposal addresses an area of research that is of high relevance in the field of aging. The studies that have been proposed will be the first to examine the potential role of signaling from lipid mediators such as the HETEs in muscle atrophy; thus the opportunity exists to make some fundamental novel discoveries in this project which may have long-term implications in improving the quality of life in the aging population.